Mammalian transcriptional activators have often been identified and characterized by transfection assays that require one or more templates to be transported and assembled in the nuclei of recipient cells. Recent efforts to understand the mechanisms of transcriptional activation on defined templates in vitro have helped define both cis-acting sequences and protein domains essential for gene regulation (Natarajan et al., 1999; Neely et al., 2002; Utley et al., 1994; Wallberg et al., 2000). However, limitations of this approach include the inability to assess contributions to transcription mediated by the trafficking of the transcription factors prior to or during chromatin assembly (Archer et al., 1992; Smith et al., 1997).
EBNA-1, encoded by Epstein-Barr virus (EBV), is a multifunctional protein essential for both EBV's extrachromosomal replication and positive and negative regulation of multiple viral promoters (Gahn et al., 1995; Lee et al., 1999; Sugden et al., 1989). EBNA-1 also positively regulates heterologous promoters when the family of repeats (FR) enhancer to which it binds in oriP is placed upstream of those promoters (Ceccarelli et al., 1998; Langle-Rouault et al., 1998; Mackey et al., 1999; Reisman et al., 1986; Wu et al., 2002). Studies of templates with or without FR microinjected into the cytoplasm or nuclei of cells that express EBNA-1 indicated that a significant contribution of EBNA-1 to the activation of transcription occurs in the cytoplasm (Langle-Rouault et al., 1998). Other studies have been interpreted to mean that EBNA-1 activates transcription on extrachromosomal but not integrated templates (Kang et al., 2001). These observations indicate that EBNA-1 likely uses multiple mechanisms to contribute to the support of transcription. If EBNA-1 can affect the transcription of integrated templates, EBNA-1 might regulate cellular genes during the latent phase of the EBV life cycle and, perhaps, in EBV-associated tumors.
EBV causes Burkitt's lymphoma (BL), which is endemic in Africa. BL is an aggressive B cell malignancy with a high proliferative rate that may be fatal within months if not treated promptly (Evens et al., 2002). Activation of the c-myc oncogene through reciprocal chromosomal translocations that juxtapose c-Myc to one of the Ig loci characterizes most BLs (Boxer et al., 2001). Additionally, many BLs carry point mutations in the p53 tumor suppressor gene or other defects in the p14ARF-MDM2-p53 pathway, or have p16INK4a genes activated by promoter methylation or homozygous deletion (Lindstrom et al., 2001; Lindstrom et al., 2002). Thus, BL involves multiple genetic events likely to promote cellular proliferation and inhibit apoptosis. In areas where BL is endemic, virtually all cases are associated with EBV (Niedobitek et al., 2001). Multiple viral genes are used by EBV to induce and maintain proliferation of infected B cells, but most of these genes are not expressed in BL tumors (Rowe et al., 1987), making it difficult to know what, if anything, EBV contributes to the survival of BL tumors.
Vectors derived from EBNA-1 are being considered for gene therapy in people (Banerjee et al., 1995; Calos, 1996; Cui et al., 2001; Franken et al., 1996; Harada et al., 2000; Phillips et al., 1999; Sclimenti et al., 1998; Stoll et al., 2001; Wohlgemuth et al., 1996). U.S. Pat. No. 4,686,186 discloses an EBV vector system which includes oriP and EBNA-1. However, EBNA-1, when overexpressed in a cell, is cytotoxic to that cell. This cytotoxicity limits its use in certain cell culture applications as well as in gene therapy. EBNA-1 may also be oncogenic (Wilson et al., 1996), further limiting its use. This increased risk of tumor development has been attributed in part to the transcriptional activation of host genes by EBNA-1 (Tsimbouri et al., 2002).
What is needed is an improved extrachromosomal vector system.